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ONCOLOGY

Comparison of molecular assays for detection and typing of human papillomavirus Christoph Koidl, MD; Michael Bozic; Ita Hadzisejdic, MD; Maja Grahovac, MD; Blazenka Grahovac, PhD; Wolfgang Kranewitter, PhD; Egon Marth, MD, PhD; Harald H. Kessler, MD OBJECTIVE: The objective of the study was to compare the performance of 3 different extraction instruments in conjunction with 4 different amplification and detection kits for detection and typing of human papillomavirus (HPV) deoxyribonucleic acid (DNA).

typing test. After extraction on the easyMAG instrument, 32 samples tested positive when the PapilloCheck with the HotStarTaq DNA polymerase was used.

RESULTS: In 31 samples, HPV DNA was detected by both the Amplicor

CONCLUSION: Together with extraction on the easyMAG instrument, the Amplicor HPV test, the LINEAR ARRAY HPV genotyping test, and the new PapilloCheck with the HotStarTaq DNA polymerase provide comparable results allowing reliable and safe HPV diagnostics in the routine laboratory. Use of alternative assays may lead to an increase of invalid and divergent HPV typing results.

HPV test and the LINEAR ARRAY HPV genotyping test in conjunction with DNA extraction on the easyMAG instrument. In another 6 samples, only low-risk types were detected with the LINEAR ARRAY HPV geno-

Key words: DNA extraction, human papillomavirus, microarray, polymerase chain reaction, typing

STUDY DESIGN: A total of 42 cervical swabs were investigated. HPV

DNA was extracted on the 3 different instruments. Each of the extracts was then amplified, and HPV DNA amplification products were detected with 4 different kits.

Cite this article as: Koidl C, Bozic M, Hadzisejdic I, et al. Comparison of molecular assays for detection and typing of human papillomavirus. Am J Obstet Gynecol 2008;199:144.e1-144.e6.

T

he human papillomavirus (HPV) has been found to be responsible for development of cervical cancer and its associated precancerous lesions.1,2 Cervical cancer is the most common female cancer in developing countries with an estimated global incidence of approximately 470,000 cases and 233,000 deaths per year.3,4 Because of organized cervical screening programs in developed countries that detect early stages of this disease, the incidence of fatal cases is significantly lower.5

Fixed-interval surveillance of women with cervical cytology screening based either on Papanicolaou staining of epithelial cells or liquid-based cytology sampled from the cervix is currently 1 of the most successful cancer screening programs. However, cervical cytology lacks sensitivity; moreover, no international consensus with regard to the overall sensitivity of classical cytology for the detection of histologically suspicious areas of the cervix has been established.6-8 Therefore, cervical cancer screening strategies

From the Molecular Diagnostics Laboratory, Institute of Hygiene, Medical University of Graz, Graz, Austria (Drs Koidl, Marth, and Kessler and Mr Bozic); the Department of Pathology, Medical Faculty University of Rijeka, Rijeka, Croatia (Drs Hadzisejdic and B. Grahovac); the Department of Dermatology and Venerology, Clinical Hospital Center Zagreb, Zagreb, Croatia (Dr M. Grahovac); and the Institute of Laboratory Medicine, General Hospital Linz, Linz, Austria (Dr Kranewitter). Received Aug. 20, 2007; revised Dec. 17, 2007; accepted March 3, 2008. Reprints: Professor Dr Harald H. Kessler, Molecular Diagnostics Laboratory, Institute of Hygiene, Medical University of Graz, Universitaetsplatz 4, A-8010 Graz, Austria. [email protected]. This study was supported in part by grants from the Austrian-Croatian Scientific and Education Cooperation Action Program and from Lambda GmbH. 0002-9378/$34.00 • © 2008 Mosby, Inc. All rights reserved. • doi: 10.1016/j.ajog.2008.03.005

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based on molecular HPV testing have been introduced. Depending on time, cost, organization, and different national screening policies, molecular HPV testing has been implemented in fixed-interval surveillance of women in recent years.9 More than 90 different HPV genotypes have been cloned and officially designated. More than 40 HPV genotypes may infect the human anogenital tract and 15 of these HPV types are classified as high-risk (HR) types for the development of cervical cancer and its precancerous lesions.10,11 For direct detection and typing of HPV in clinical samples, molecular assays have been introduced recently. Currently 3 commercially available assays, the Hybrid Capture 2 (Digene Corp, Gaithersburg, MD), the Amplicor HPV test (Roche Molecular Systems, Inc, Branchburg, NJ), and the LINEAR ARRAY HPV genotyping test (Roche) assays have been evaluated extensively and found to be appropriate for use in primary screening and triage.12-15 In the present study, 42 cervical swabs were investigated. The performance of 3

Oncology

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FIGURE 1

Study flow diagram

HPV DNA was extracted out of each sample with 3 different instruments. For amplification and detection of HPV DNA, 4 different kits were used. Koidl. Comparison of molecular assays in human papillomavirus. Am J Obstet Gynecol 2008.

different methods for extraction of HPV DNA in conjunction with 4 different methods for PCR amplification and detection of amplification products were compared.

HPV amplification and detection assays, yielding a total of 504 results (Figure 1). Results obtained by different assay were compared.

Sample preparation

M ATERIALS AND M ETHODS Samples A total of 42 cervical swabs obtained from women with persistent Papanicolaou III D status in cervical cytology were studied. Cervical swabs were taken with Cervex-Brush cervical cell sampler (Rovers Medical Devices B.V., Oss, The Netherlands) and collected in 20 mL ThinPrep PreservCyt solution transport medium (Cytec Corp, Boxborough, MA) according to the manufacturer’s instructions. Samples were stored at 4°C until analysis within 3 weeks after collection.

Study design HPV deoxyribonucleic acid (DNA) was extracted from 42 cervical swabs with 3 different commercially available methods yielding a total of 126 extracts. Extracts were investigated with 4 different

Automated HPV DNA extraction was performed on each of 3 commercially available sample preparation instruments: the easyMAG (bioMerieux sa, Marcy l’Etoile, France), the MagNA Pure LC (Roche Applied Science, Mannheim, Germany), and the BioRobot EZ1 workstation (QIAGEN, Hamburg GmbH, Hamburg, Germany). When HPV DNA was extracted on the easyMAG, the NucliSens easyMAG accessory products (bioMerieux) were used. A total of 400 ␮L of the transport medium were pipetted into the easyMAG reaction tube. Samples were prepared with the automated extraction protocol program Generic 1.0.6 (bioMerieux) including an additional manual external lysis step (4 times stirring of the sample with a pipette). The elution volume was set to be 110 ␮L.

Research

Prior to automated HPV DNA extraction on the MagNA Pure LC, 400 ␮L of the sample transport medium were centrifuged with 12,000 rpm for 5 minutes at room temperature. After discarding the supernatant, the pellet was resuspended in 190 ␮L of tissue lysis buffer (Roche Applied Science) followed by addition of 10 ␮L of Proteinase K (Roche Applied Science). After incubation on a mixing platform for 3 hours at 56°C, the sample was transferred into the MagNA Pure LC reaction tube. On the MagNA Pure LC, HPV DNA was extracted with the MagNA Pure DNA Isolation Kit I (Roche Applied Science). The DNA I high-performance external lysis program (Roche Applied Science) was used. The elution volume was set to be 200 ␮L. Prior to automated HPV DNA extraction on the BioRobot EZ1 workstation, 200 ␮L of the sample transport medium were centrifuged with 12000 rpm for 5 minutes at room temperature. After discarding the supernatant, the pellet was resuspended in 190 ␮l buffer G2 (QIAGEN) followed by the addition of 10 ␮L Proteinase K (QIAGEN). After incubation on a mixing platform for 3 hours at 56°C, the sample was transferred into the BioRobot EZ1 reaction tube. On the BioRobot EZ1 workstation, HPV DNA was extracted with the EZ1 DNA tissue kit (QIAGEN) using the EZ1 DNA tissue card (QIAGEN). The elution volume was set to be 100 ␮L.

Amplification and detection of HPV DNA Amplification and detection of HPV DNA were performed with each of 4 assays: the Amplicor HPV test (Roche Applied Science), the LINEAR ARRAY HPV genotyping test (Roche Applied Science), the PapilloCheck (Greiner BioOne GmbH, Frickenhausen, Germany) with AmpliTaq Gold (Applied Biosystems, Foster City, CA), and the PapilloCheck (Greiner Bio-One) with HotStarTaq DNA polymerase (QIAGEN). The Amplicor HPV test is designed for qualitative detection of HPV DNA. It provides detection of HR HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68 without typing. When HPV DNA was amplified with the Amplicor HPV

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Oncology

test, the Gene Amp PCR System 9700 (Applied Biosystems) was used according to the manufacturer’s instructions. Fully automated hybridization and detection of HPV DNA was done on the BEP III (Dade Behring Marburg GmbH, Marburg, Germany). The LINEAR ARRAY HPV genotyping test is designed for qualitative detection and typing of HPV types 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, CP6108, and IS39. When HPV DNA was amplified with the LINEAR ARRAY HPV genotyping test, the Gene Amp PCR System 9700 was used according to the manufacturer’s instructions. Fully automated hybridization and detection of HPV DNA was done on the ProfiBlot 48 (Tecan Trading AG, Zurich, Switzerland). The PapilloCheck is designed for qualitative detection and typing of HPV types 6, 11, 16, 18, 31, 33, 35, 39, 40, 42, 43, 44, 45, 51, 52, 53, 56, 58, 59, 66, 68, 70, 73, and 82. The PapilloCheck master mix does not include the DNA polymerase. In this study, either the AmpliTaq Gold or the HotStarTaq DNA polymerase was added to the master mix. When the HPV DNA was amplified with the PapilloCheck, the Gene Amp PCR System 9700 thermocycler was used according to the manufacturer’s instructions. After hybridization, analysis of the PapilloCheck DNA chip was performed on the CheckScanner (Greiner Bio-One) using the CheckReport (Greiner BioOne) analysis software according to the package inserts. Both the Amplicor HPV test and the LINEAR ARRAY HPV genotyping test include a primer pair for amplification and detection of the human beta-globin gene as heterologous internal control for cell adequacy, extraction, and amplification. The PapilloCheck includes both a primer pair for amplification and detection of the human ADAT1 gene as heterologous control for cell adequacy, extraction, and amplification and another primer pair for the amplification and detection of an artificial control template as an additional control for amplification. If any of the internal controls were not 144.e3

www.AJOG.org detected, the result was considered as invalid.

R ESULTS Of 42 cervical swabs, HPV DNA was not detected in 5 samples with any of the tests used. In 31 samples, HPV DNA was detected by the Amplicor HPV test and the LINEAR ARRAY HPV genotyping test, both in conjunction with DNA extraction on the easyMAG instrument. All of these samples contained either only HR HPV types or a combination of HR and low-risk (LR) HPV types. In another 6 samples, only LR HPV types were found with the LINEAR ARRAY HPV genotyping test. With both of these assays, the heterologous internal control was consistently detected. When the Amplicor HPV test was used in conjunction with DNA extraction on the MagNA Pure LC instrument, 26 of 37 samples with HR HPV types tested positive. One of the positives contained only LR HPV types and was thus considered to be false positive. Three samples tested negative and 8 gave an invalid result. When the Amplicor HPV test was used in conjunction with DNA extraction on the BioRobot EZ1 workstation, 14 of 37 samples with HR HPV types tested positive. One of the positive samples contained only LR HPV types and was thus considered to be false positive. Four samples tested negative. Two of the samples contained an HR HPV type and were thus considered to be false negative. The remaining 19 samples gave an invalid result. When the LINEAR ARRAY HPV genotyping test was performed for HPV DNA amplification and typing after DNA extraction with the easyMAG, a single or more HPV types were found in 37 samples. The heterologous internal control was consistently detected in all samples. After DNA extraction with the MagNA Pure LC instrument, 29 samples with a single or more HPV types, 2 false negatives and 6 invalid results were obtained. After DNA extraction with the BioRobot EZ1 workstation, 11 samples with a single or more HPV types, 9 false negatives, and 17 invalid results were obtained.

American Journal of Obstetrics & Gynecology AUGUST 2008

FIGURE 2

Number of invalid results when using different extraction instruments

Number of invalid results when using different extraction instruments in conjunction with kits for qualitative detection and typing of HPV types. x-axis, kits used in this study; y-axis, extraction instruments used in this study. Koidl. Comparison of molecular assays in human papillomavirus. Am J Obstet Gynecol 2008.

With the LINEAR ARRAY HPV genotyping test, 2 samples were found to contain only bands CP6108 and HPV 62, respectively. Because these HPV types are not detectable with the PapilloCheck assay, valid negative results for those samples obtained by the PapilloCheck assay were referred to as true negative. When the PapilloCheck with the AmpliTaq Gold DNA polymerase in the master mix was performed for HPV DNA amplification and typing after DNA extraction with the easyMAG, a single or more HPV types were obtained in 30 samples. Of the remaining 7 samples, 2 were true negative (see aforementioned text), 3 were false negative, and 2 gave invalid results. After DNA extraction with the MagNA Pure LC instrument, a single or more HPV types were obtained in 12 samples. Of the remaining 25 samples, 1 was false negative and 24 gave invalid results. After DNA extraction with the BioRobot EZ1 workstation, a single or more HPV types were obtained in 20 samples. Of the remaining 17 samples, 1 was true negative (see aforementioned text), 3 were false negative, and 13 gave invalid results. When the PapilloCheck with the HotStarTaq DNA polymerase in the master mix was performed for HPV DNA amplification and typing after DNA extraction with the easyMAG, a single or more

Results obtained with HPV assays for qualitative detection and typing of HPV types easyMAG Sample no.

MagNA Pure LC

BioRobot EZ1

Linear Array

PapilloCheck/ AmpliTaq

PapilloCheck/ HotStarTaq

Linear Array

PapilloCheck/ AmpliTaq

PapilloCheck/ HotStarTaq

Linear Array

PapilloCheck/ AmpliTaq

PapilloCheck/ HotStarTaq

1

18

18, 45

18, 45

18

18, 45

18, 45

18

18

18, 45, 70

2

16, 33, 42, 53, 59, 66

16, 42, 43, 59

16, 33, 42, 53, 56, 59

16, 42, 53, 59, 66

42, 53, 59

16, 42, 53, 56, 59

Invalid

42, 53

16, 42, 53, 56, 59, 66, 70, 73

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TABLE

............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

3

66

False negative

False negative

False negative

False negative

False negative

Invalid

False negative

False negative

4

66, 56

56, 66

54, 55 56, 66

66

Invalid

66

66

66

44, 45, 56, 59, 66, 70, 73, 82

............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

5

16

Invalid

Invalid

Invalid

Invalid

Invalid

16

Invalid

False negative

6

31

31

31

31

Invalid

45

31

31

31

7

16

16

16

16

16, 45

16, 56

16

16

16, 56

8

59

59

59

59

Invalid

Invalid

False negative

59

59, 73

9

31

31

31

31

Invalid

31

Invalid

Invalid

31

10

18, 52

18, 52

18, 52

18, 52

45, 52

18, 52

18, 52

18, 52

18, 52

11

33, 52

33

33

33

Invalid

Invalid

33

33

33

12

16

16

16

16

Invalid

45

16

False negative

16

13

70

False negative

False negative

70

Invalid

Invalid

70

Invalid

False negative

14

CP6108

True negative

True negative

False negative

Invalid

Invalid

CP6108

Invalid

True negative

15

16, 31, 52, 56

16, 56

16, 31, 56, 66

56

Invalid

Invalid

False negative

Invalid

16, 56

16

6, 66, 84

66

56, 59, 66

66, 84

66

56, 66

False negative

Invalid

66

17

16

16

16

Invalid

Invalid

Invalid

False negative

Invalid

Invalid

18

16, 31

16, 31

16, 31

31

Invalid

31

False negative

31

16, 31, 66

19

53, 54, 61, 66, 82, 84

56, 66, 82

53, 56, 66, 82

53, 54, 61, 66, 82, 84

45, 66, 82

53, 56, 66, 82

66

Invalid

66, 82

20

52, CP6108

Invalid

52

Invalid

Invalid

Invalid

False negative

Invalid

52

21

39

39

39

39

Invalid

Invalid

False negative

Invalid

39

22

51

51

51

51

51

51

False negative

51

51

23

31, 62, 66

66

31, 56, 66

31, 62, 66

66

31, 56, 66

Invalid

Invalid

56, 66

24

66

66

56, 66

Invalid

Invalid

66

Invalid

66

56, 66

25

16, 31, 42, 59, 66

42, 59

16, 31, 42, 59, 66

59

Invalid

42, 59

Invalid

42, 59

............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ a a a ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................ ............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

31, 42, 59

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Continued on page 144.e5.

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144.e5

16, 81

16

33, 52, 81

31, 62, 81

58, 56

16, 31, 52, 59

16

62

16, 51, 66

52, 62

51, 53, 56, 58

56, 61, 66, 68, 73

26

27

28

29

30

31

32

33

34

35

36

37

16

PapilloCheck/ AmpliTaq 16

PapilloCheck/ HotStarTaq

PapilloCheck/ AmpliTaq Invalid

Invalid

Invalid

Invalid

Invalid

Linear Array Invalid

Invalid

33

31, 81

56

MagNA Pure LC

45

PapilloCheck/ HotStarTaq Invalid

Linear Array

BioRobot EZ1

16

PapilloCheck/ AmpliTaq 16

PapilloCheck/ HotStarTaq

16

33, 51

Invalid

False negative

Invalid

Invalid

16

False negative

16

33

31

31

False negative

31

31

56

Invalid

Invalid

Invalid

56

Invalid

16

Invalid

52

56

56, 66

16

16

62

16, 51

52

53, 56, 58

56, 61, 66, 68, 73

True negative

16, 51, 66, 82

16, 31, 52

16, 82

Invalid

16, 45

Invalid

Invalid

Invalid

16

16, 31

16

Invalid

Invalid

Invalid

True negative

Invalid

16

Invalid

16, 51, 66, 82

Invalid

American Journal of Obstetrics & Gynecology AUGUST 2008

52

53, 56

52

53, 56

Invalid

Invalid

52

56

52

56

56, 66

Both HPV 6108 and HPV 62 not detectable by the PapilloCheck.

Koidl. Comparison of molecular assays in human papillomavirus. Am J Obstet Gynecol 2008.

a

45, 56, 66

Invalid

56, 66

56, 66

............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

56

......................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

56

......................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

52, 31

......................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

16, 51

......................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

True negative

...................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................... a a a

False negative

......................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

16, 31

......................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

56

......................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

31

............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

33

............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

16

............................................................................................................................................................................................................................................................................................................................................................................................................................................................................................................

Linear Array

Sample no.

easyMAG

Continued from page 144.e4.

Results obtained with HPV assays for qualitative detection and typing of HPV types

TABLE

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HPV types were obtained in 32 samples. Of the remaining 5 samples, 2 were true negative (see aforementioned text), 2 were false negative, and one gave an invalid result. After DNA extraction with the MagNA Pure LC instrument, a single or more HPV types were obtained in 22 samples. Of the remaining 15 samples, 2 were false negative and 13 gave invalid results. After DNA extraction with the BioRobot EZ1 workstation, a single or more HPV types were obtained in 30 samples. Of the remaining 7 samples, 1 was true negative (see aforementioned text), 3 were false negative, and 3 gave invalid results. Numbers of invalid results when using different extraction instruments in conjunction with kits for qualitative detection and typing of HPV types are shown in Figure 2. Detailed HPV typing results are demonstrated in the Table. When 8 samples were analyzed in parallel, DNA extraction including manual pretreatment of samples required 50 min with the easyMAG. When alternative extraction instruments were used, 70 minutes were required with the MagNA Pure LC instrument and 230 minutes with the BioRobot EZ1 workstation. For amplification and detection, the Amplicor HPV test required 300 minutes, the LINEAR ARRAY HPV genotyping test 340 minutes, and the PapilloCheck 210 minutes.

C OMMENT

Chronic infection with HPV is responsible for development of cervical cancer and its associated precancerous lesions.1,2 In addition to classical cervical screening with Papanicolaou staining or liquid-based cytology, molecular detection of HPV DNA has been implemented in fixed-interval surveillance of women in recent years.9 For detection and typing of HPV DNA, currently 3 commercially available assays are predominantly used for cervical surveillance and triage.12-15 In this study, the performance of 3 different methods for extraction of HPV in conjunction with 4 different methods for PCR amplification and detection of amplification products were compared. The rationale of this study was to identify differences in the outcome of results.

Oncology

www.AJOG.org When the Amplicor HPV test and the LINEAR ARRAY HPV genotyping test were used in conjunction with DNA extraction on the easyMAG instrument, concordant results were obtained for all samples. With the Amplicor assay in conjunction with DNA extraction on the MagNA Pure LC instrument, 1 of the LR HPV DNA samples was found to be positive. This result was considered to be false positive and may be explained by carryover contamination because of the open mode system of this extraction instrument. In contrast, both of the alternative extraction instruments used in this study use closed mode systems to minimize contamination risk during sample preparation. When the Amplicor HPV test and the LINEAR ARRAY HPV genotyping test were used in conjunction with DNA extraction on the easyMAG instrument, no invalid results were found in all samples tested. With alternative DNA extraction instruments, both an increased number of invalid results and false-negative samples were found, emphasizing the need of inclusion of internal controls in molecular assays in general and the weakness of alternative extraction instruments used in this study.16 When the 3 HPV genotyping assays used in this study were compared, HPV typing results were found to be inhomogeneous in infections with multiple HPV types. The performance of the PapilloCheck with the HotStarTaq DNA Polymerase in the master mix was found to be superior to that with the AmpliTaq Gold in the master mix. For the laboratory workflow, sample processing time is a major issue. Time required for sample preparation includ-

ing manual steps and automated sample processing on the easyMAG instrument was found to be superior to that with both the MagNA Pure LC instrument and the BioRobot EZ1 workstation. For HPV amplification and detection, the PapilloCheck assay showed the best time efficiency. In conclusion, the Amplicor HPV test, the LINEAR ARRAY HPV genotyping test, and the new PapilloCheck with the HotStarTaq DNA polymerase provide comparable results when performed after extraction on the easyMAG instrument, allowing reliable and safe HPV diagnostics in the routine laboratory. Use of alternative assays may lead to an increase of invalid results and divergent f HPV typing results. ACKNOWLEDGMENT The authors gratefully acknowledge Michael Schleichert for stimulating discussions and Jörg Berg for technical support.

REFERENCES 1. Bosch FX, Lorincz A, Munoz N, Meijer CJ, Shah KV. The causal relation between human papillomavirus and cervical cancer. J Clin Pathol 2002;55:244-65. 2. Walboomers JM, Jacobs MV, Manos MM, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999;189:1-3. 3. Einstein MH, Goldberg GL. Human papillomavirus and cervical neoplasia. Cancer Invest 2002;20:1080-5. 4. Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan 2000. Int J Cancer 2001;94:153-6. 5. Parkin DM, Pisani P, Ferlay J. Estimates of the worldwide incidence of 25 major cancers in 1990. Int J Cancer 1999;80:827-41. 6. Fahey MT, Irwig L, Macaskill P. Meta-analysis of Pap test accuracy. Am J Epidemiol 1995; 141:680-9.

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7. Bernstein SJ, Sanchez-Ramos L, Ndubisi B. Liquid-based cervical cytologic smear study and conventional Papanicolaou smears: a metaanalysis of prospective studies comparing cytologic sample diagnosis and sample adequacy. Am J Obstet Gynecol 2001;185: 308-17. 8. Castle PE, Sadorra M, Garcia F, Holladay EB, Kornegay J. A pilot study of a commercialized human papillomavirus (HPV) genotyping assay: comparison of HPV risk group to cytology and histology. J Clin Microbiol 2006;44:3915-7. 9. Berkhof J, de Bruijne MC, Zielinski GD, et al. Evaluation of cervical screening strategies with adjunct high-risk human papillomavirus testing for women with borderline or mild dyscakaryosis. Int J Cancer 2006;118:1759-68. 10. Munoz N, Bosch FX, de Sanjose S, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348:518-27. 11. zur Hausen H. Papillomaviruses and cancer: From basic studies to clinical application. Nat Rev Cancer 2002;2:342-50. 12. Giuliani L, Coletti A, Syrjanen K, Favalli C, Ciotti M. Comparison of DNA sequencing and Roche Linear Array in human papillomavirus (HPV) genotyping. Anticancer Res 2006; 26:3939-41. 13. Monsonego J, Bohbot JM, Pollini G, et al. Performance of the Roche AMPLICOR human papillomavirus (HPV) test in prediction of cervical intraepithelial neoplasia (CIN) in women with abnormal PAP smear. Gynecol Oncol 2005; 99:160-8. 14. Sandri MT, Lentati P, Benini E, et al. Comparison of the Digene HC2 assay and the Roche AMPLICOR human papillomavirus (HPV) test for detection of high-risk HPV genotypes in cervical samples. J Clin Microbiol 2006;44:2141-6. 15. Stevens MP, Rudland E, Garland SM, Tabrizi SN. Assessment of MagNA pure LC extraction system for detection of human papillomavirus (HPV) DNA in PreservCyt samples by the Roche AMPLICOR and LINEAR ARRAY HPV test. J Clin Microbiol 2006;44:2428-33. 16. Stöcher M, Leb V, Holzl G, Berg J. A simple approach to the generation of heterologous competitive internal controls for real-time PCR assays on the LightCycler. J Clin Virol 2002; 25:47-53.

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